RE: [Histonet] SMC Immuno and Elastin combo?
Nancy, The red of the classic VG counterstain stains the collagen. Muscle will appear yellow. If the yellow is obscuring the brown DAB then after VG staining wash the slides in water. This will remove the picric acid staining leaving the muscle brown. Since Nova red is red (I assume since I have not had cause to use it - I'm a DAB man!), I would use Picro Indigo carmine or Picro-Light green. Picro-indigo Solution (Ortiz-Hidalgo C "Abelardo Gallego(1879-1930)and his contributions to histotechnology: The Gallego stains" Acta histochemica1 13(2011):189-193): Picric acid (saturated solution)100ml Indigo carmine 0.25mg Distilled water 15ml Whippy's Elastic Stain (Whippy P "A Stain to Try - Whippy's Elastic Stain" Histograph Jan 2008:7): Light Green Stock Solution Light Green 2 gm Distilled water 100 ml Glacial Acetic Acid 2 ml Place Light Green and distilled water into a conical flask and mix well. Add Acetic acid and mix. Filter and pour into stock bottle. Light Green - Picrate (Whippy's) Solution Saturated aqueous picric acid 10 ml Light Green stock solution5 drops Pour Picric Acid into a conical flask. Add Light Green solution, stir until dissolved. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager & Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Thomas, Nancy Sent: Thursday, 14 March 2013 3:33 AM To: 'Patricia Bourassa'; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] SMC Immuno and Elastin combo? Patti, The immunostaining probably was not wiped out, rather just covered up with the red color of the VG counterstain. You might try a chromogen that expresses in a different color. Or, you might leave off the van gieson part of the EVG (staining only the elastic tissue) and pick a counterstain of another color. Basically, you just need to make sure that none of the stains used have the same color outcome. I have done this combo in the past. Never really finished the project because the issue I had was that the lizard tissue I was working with just would not stay on the slide no matter what I tried. But, I do remember that I treated them as two separate stains. I did the actin staining first, rinsed it well, then started in on the elastin stain. You probably have a protocol for each of these and you just need to combine them. Nancy Thomas Stowers Institute -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Patricia Bourassa Sent: Wednesday, March 13, 2013 10:58 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] SMC Immuno and Elastin combo? Hello, I've read some papers which reference the combination of smooth muscle cell actin immunostaining with a Verhoeff's elastin counterstain. Has anyone ever done this and if so, do you have protocol you'd be willing to share? I'm staining mouse and rat lung and have a SMC protocol working that uses an Abcam antibody and NovaRed chromagen (peroxidase). I've also done EVG staining so I'm familiar with the general process. The combination is what I'm not sure of - I gave it a try for the heck of it and wiped out the immunostain. We've just started looking at this, so nothing is set in stone and I'd be grateful for any advice/ procedural help. Thanks in advance! Patti Bourassa Karos Pharmaceuticals -- *Patti Bourassa Senior Scientist Karos Pharmaceuticals (203) 535-0540, ext 207* ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet * This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expres
Re: [Histonet] SMC Immuno and Elastin combo?
Patti, You're using a mouse monoclonal antibody on mouse tissue, so be aware that the secondary link antibody will bind to any mouse IgG it finds in the tissue (normal plasma cells, IgG in normal sera present inside blood vessels, or as infiltrating interstitial normal sera, etc.), not just to the primary anti-Actin antibody. You may have to use a mouse-on-mouse kit for the Actin staining portion, if you see too much nonspecific background in your tissue. Jan Shivers UMN Vet Diag Lab On Wed, Mar 13, 2013 at 10:58 AM, Patricia Bourassa < pboura...@karospharma.com> wrote: > Hello, > > I've read some papers which reference the combination of smooth muscle cell > actin immunostaining with a Verhoeff's elastin counterstain. > > Has anyone ever done this and if so, do you have protocol you'd be willing > to share? > > I'm staining mouse and rat lung and have a SMC protocol working that uses > an Abcam antibody and NovaRed chromagen (peroxidase). I've also done EVG > staining so I'm familiar with the general process. The combination is what > I'm not sure of - I gave it a try for the heck of it and wiped out the > immunostain. > > We've just started looking at this, so nothing is set in stone and I'd be > grateful for any advice/ procedural help. > > Thanks in advance! > > Patti Bourassa > Karos Pharmaceuticals > -- > *Patti Bourassa > Senior Scientist > Karos Pharmaceuticals > (203) 535-0540, ext 207* > ___ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] SMC Immuno and Elastin combo?
Patti, The immunostaining probably was not wiped out, rather just covered up with the red color of the VG counterstain. You might try a chromogen that expresses in a different color. Or, you might leave off the van gieson part of the EVG (staining only the elastic tissue) and pick a counterstain of another color. Basically, you just need to make sure that none of the stains used have the same color outcome. I have done this combo in the past. Never really finished the project because the issue I had was that the lizard tissue I was working with just would not stay on the slide no matter what I tried. But, I do remember that I treated them as two separate stains. I did the actin staining first, rinsed it well, then started in on the elastin stain. You probably have a protocol for each of these and you just need to combine them. Nancy Thomas Stowers Institute -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Patricia Bourassa Sent: Wednesday, March 13, 2013 10:58 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] SMC Immuno and Elastin combo? Hello, I've read some papers which reference the combination of smooth muscle cell actin immunostaining with a Verhoeff's elastin counterstain. Has anyone ever done this and if so, do you have protocol you'd be willing to share? I'm staining mouse and rat lung and have a SMC protocol working that uses an Abcam antibody and NovaRed chromagen (peroxidase). I've also done EVG staining so I'm familiar with the general process. The combination is what I'm not sure of - I gave it a try for the heck of it and wiped out the immunostain. We've just started looking at this, so nothing is set in stone and I'd be grateful for any advice/ procedural help. Thanks in advance! Patti Bourassa Karos Pharmaceuticals -- *Patti Bourassa Senior Scientist Karos Pharmaceuticals (203) 535-0540, ext 207* ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] SMC Immuno and Elastin combo?
Hello, I've read some papers which reference the combination of smooth muscle cell actin immunostaining with a Verhoeff's elastin counterstain. Has anyone ever done this and if so, do you have protocol you'd be willing to share? I'm staining mouse and rat lung and have a SMC protocol working that uses an Abcam antibody and NovaRed chromagen (peroxidase). I've also done EVG staining so I'm familiar with the general process. The combination is what I'm not sure of - I gave it a try for the heck of it and wiped out the immunostain. We've just started looking at this, so nothing is set in stone and I'd be grateful for any advice/ procedural help. Thanks in advance! Patti Bourassa Karos Pharmaceuticals -- *Patti Bourassa Senior Scientist Karos Pharmaceuticals (203) 535-0540, ext 207* ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
